Location and sequence conservation of Tyr199 and Tyr309 in human PANX1.

A, The Tyr199 and Tyr309 of hPANX1 are located close to the side tunnel. Two adjacent subunits are shown in semi-transparent surface representation. The Tyr199 and Tyr309 are shown in stick representation. The green spheres indicate the pathway of the side tunnel. B, Sequence alignment of PANX1 orthologs, human PANX2 and PANX3. Tyr198 and Tyr309 are highlighted using a red circle. Secondary structural features are shown on the top.

Commercially available phospho-PANX1 antibodies (anti-PANX1-pY198 and anti-PANX1-pY308) do not recognize PANX1.

A, A schematic figure showing different mSrc mutants used in the study. Mouse Src WT undergoes a dynamic equilibrium between the inactive and active states that depends on the phosphorylation status of Tyr529. The Y529F mutation renders Src constitutively active due to the disruption of the autoinhibitory interaction between the SH2 domain and the C-terminal tail(34). The K297M is catalytically incompetent as the mutation abolishes the ATP-binding capability of mSrc(42). B, Human PANX1 (WT, Y199F or Y309F) are co-expressed with mSrc (WT or Y529F) in HEK293T cells. The cell lysates are analyzed by SDS gel and blotted with anti-PANX1-pY198 (top) and anti-PANX1-pY308 (middle) antibodies. The in-gel fluorescence of GFP and mCherry signal are shown at the bottom. The positions of the signals detected by anti-PANX1-pY198 and anti-PANX1-pY308 antibodies do not match the position of the GFP fluorescence signal, indicating that the two antibodies are not specific for PANX1.

Figure 2—source data 1: Uncropped immunoblots for Figure 2B.

Figure 2—source data 2: Raw image for anti-PANX1-pY198 immunoblot in Figure 2B.

Figure 2—source data 3: Raw image for anti-PANX1-pY308 immunoblot in Figure 2B.

Figure 2—source data 4: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 2B.

The anti-PANX1-pY198 and anti-PANX1-pY308 are not specific to hPANX1 in vitro.

A, In vitro phosphorylation of hPANX1-GFP WT by active human Src-GST protein. The leftmost panel showed the in-gel fluorescence of GFP overlaid with protein marker. The other panels (from left to right) represent the western blot signal of anti-PANX1, anti-pY100, anti-Src, anti-PANX1-PANX1-pY198, and anti-PANX1-pY308 antibodies, respectively. B, In vitro phosphorylation of hPANX1-GFP WT, L306A, K307A, V308A, Y309F, E310A, I311A, or L312A mutants. The left panel showed the in-gel fluorescence of GFP overlaid with protein marker. The right panel showed the western blot signal of anti-PANX1-pY308 antibody. Only hPANX1 WT lane showed western blot signal. C, In vitro phosphorylation of hPANX1-GFP WT, Y309A, or Y309F mutants. The left panel showed the in-gel fluorescence of GFP overlaid with protein marker (red). The right panel showed the western blot signal of anti-PANX1-pY308 antibody. The Y309A mutant rendered PANX1 prone to oligomerization in our SDS gel experiment, but not for Y309F. D, Whole cell lysate of HEK293T cells transiently transfected with hPANX1 WT or Y309F. Left panel showed the in-gel fluorescence of GFP overlaid with protein marker. Middle and right panels represent western blot result using anti-PANX1 and anti-PANX1-pY308 antibody, respectively. The location where PANX1 is expected to migrate in the SDS-PAGE gel is indicated. Of note, visualizing such a band requires a very long exposure, which resulted in a large number of non-specific bands.

Figure 3—source data 1: Uncropped immunoblots for Figure 3.

Figure 3—source data 2: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 3A.

Figure 3—source data 3: Raw image for anti-PANX1 immunoblot in Figure 3A.

Figure 3—source data 4: Raw image for anti-pY100 immunoblot in Figure 3A.

Figure 3—source data 5: Raw image for anti-Src immunoblot in Figure 3A.

Figure 3—source data 6: Raw image for anti-PANX1-pY198 immunoblot in Figure 3A.

Figure 3—source data 7: Raw image for anti-PANX1-pY308 immunoblot in Figure 3A.

Figure 3—source data 8: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 3B,C.

Figure 3—source data 9: Raw image for anti-PANX1-pY308 immunoblot in Figure 3B,C.

Figure 3—source data 10: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 3D.

Figure 3—source data 11: Raw image for anti-PANX1 immunoblot in Figure 3D.

Figure 3—source data 12: Raw image for anti-PANX1-pY308 immunoblot in Figure 3D.

Human PANX1 is not phosphorylated by mSrc when expressed in HEK293T cells.

A, The Phos-tag gel result of human PANX1 co-expressed with different mSrc mutants. The GFP and mCherry fluorescence signal were overlaid to generate the image. The mSrc kinase of different phosphorylation status are labeled. In all conditions, hPANX1 migrated as a single band. B, Analysis of the same cell lysate sample in B using regular SDS gel. Western blot experiment was conducted using anti-PANX1 (top) and anti-Src (middle) antibodies. The anti-PANX1 antibody detected a non-specific band at 100 kDa from the whole-cell lysate with unknown identity. The in-gel fluorescence for GFP and mCherry is shown at the bottom where the position of mSrc and PANX1 protein could be compared with the western blot signal.

Figure 4—source data 1: Uncropped immunoblots for Figure 4.

Figure 4—source data 2: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 4A.

Figure 4—source data 3: Raw image for anti-PANX1 immunoblot in Figure 4B.

Figure 4—source data 4: Raw image for anti-Src immunoblot in Figure 4B.

Figure 4—source data 5: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 4B.

Human PANX1 is not phosphorylated by Src when expressed in Neuro2A cells.

A, A regular SDS gel showing the GFP and mCherry fluorescence signal of hPANX1 and mSrc co-expressed in Neuro2A cells. For each condition containing PANX1, PNGase F treatment is performed to de-glycosylate the protein. B, A PhosTag gel result using the same samples analyzed in A.

Figure 5—source data 1: Uncropped immunoblots for Figure 5.

Figure 5—source data 2: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 5A.

Figure 5—source data 3: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 5B.

Mouse PANX1 is not phosphorylated by Src when expressed in HEK293T cells.

A, A Phos-tag gel result of mPANX1 co-expressed with different mSrc mutants. The GFP and mCherry fluorescence signal are overlaid to generate the image. The mSrc kinase of different phosphorylation status are labeled. In all the conditions, mouse PANX1 migrates to as a single band. B, Analysis of the same cell lysate sample in B using regular SDS-PAGE gel. Western blot experiment is conducted using anti-PANX1 (top) and anti-Src (middle) antibodies. The in-gel fluorescence for GFP and mCherry is shown at the bottom where the position of mSrc and mPANX1 protein could be compared with the western blot signal.

Figure 6—source data 1: Uncropped immunoblots for Figure 6.

Figure 6—source data 2: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 6A.

Figure 6—source data 3: Raw image for anti-PANX1 immunoblot in Figure 6B.

Figure 6—source data 4: Raw image for anti-Src immunoblot in Figure 6B.

Figure 6—source data 5: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 6B.

The PANX1 current remains unchanged in the presence of active mSrc.

A, Representative fluorescent images of HEK293T cells transfected with hPANX1-GFP and mSrc-Y529F-mCherry. Cells that show both green (GFP) and red (mCherry) fluorescence are selected for whole-cell patch-clamp analysis. B, The CBX sensitive whole-cell current of hPANX1-GFP and hPANX1+mSrc-Y529F-mCherry. Two-way ANOVA for repeated measurements was conducted to compare the hPANX1-GFP and hPANX1+mSrc-Y529F-mCherry. The analysis revealed a non-significant difference between the two groups (F(1, 182)= 0.07286, P = 0.7875). Data are presented as mean ± SEM.

The hPANX1 cannot be detected by anti-PANX1-pY198 and anti-PANX1-pY308 irrespective of C-terminal GFP tag.

Human PANX1 w-or w/o C-terminal GFP tag were co-expressed with mSrc-Y529F in HEK293T cells. The cell lysates were analyzed by SDS gel and blotted with anti-PANX1-pY198, anti-PANX1-pY308, anti-PANX1, and anti-Src antibodies. The in-gel fluorescence of GFP and mCherry signal were shown at the bottom. The positions of the signals detected by anti-PANX1-pY198 and anti-PANX1-pY308 antibodies did not match the position of the anti-PANX1 western blot signal and GFP fluorescence signal.

Dephosphorylation of mSrc protein by lambda protein phosphotase (λ-PP).

Phos-tag gel showed that mSrc-mCherry WT, Y529F, and K297M protein had different phosphorylation status. Phosphorylated mSrc could be converted to the non-phosphorylated form by λ-PP.

Figure 4—figure supplement 1—source data 1: Uncropped immunoblots for Figure 4— figure supplement 1.

Figure 4—figure supplement 1—source data 2: Raw image for in-gel fluorescence (mCherry) in Figure 4—figure supplement 1 (upper panel).

Figure 4—figure supplement 1—source data 3: Raw image for in-gel fluorescence (mCherry) in Figure 4—figure supplement 1 (lower panel).

Wild-type hPANX1 without C-terminal GFP tag is not phosphorylated my mSrc.

Human PANX1 with/without C-terminal GFP tag is co-expressed with Src-mCherry WT, Y529F, or K297M in HEK293T cells. Cell lysate was subjected to PNGase F de-glycosylation prior to analysis on SDS-PAGE gel (upper and middle panels) or Phos-tag gel (bottom panel). Upper panel showed the GFP and mCherry fluorescence signal in SDS-PAGE gel. Middle panel showed the western blot result of anti-PANX1 antibody blot in SDS-PAGE gel. Bottom panel showed the western blot result of anti-PANX1 antibody blot in Phos-tag gel.

Figure 4—figure supplement 2—source data 1: Uncropped immunoblots for Figure 4— figure supplement 2.

Figure 4—figure supplement 2—source data 2: Raw image for in-gel fluorescence (GFP and mCherry) in Figure 4—figure supplement 2 (upper panel).

Figure 4—figure supplement 2—source data 3: Raw image for anti-PANX1 immunoblot in Figure 4—figure supplement 2 (middle panel).

Figure 4—figure supplement 2—source data 4: Raw image for anti-PANX1 immunoblot in Figure 4—figure supplement 2 (lower panel).

Figure 4–figure supplement 3–—figure supplement 3—source data 1: LC-MS/MS data for WT human PANX1 with or without co-expressing Src-Y529F mutant [Dataset]. Dryad. https://doi.org/10.5061/dryad.4tmpg4fh7

Coverage map of LC-MS/MS analysis of purified hPANX1-GFP.

A, The sequence coverage of hPANX1 expressed alone. B, The sequence coverage of hPANX1 expressed with the constitutively active mSrc Y529F. Of note, both Tyr199 and Tyr309 are covered by LC-MS/MS spectra.

Representative LC-MS/MS spectra containing the Tyr199 of hPANX1.

A, The raw spectrum of (K)YPIVEQYLKTK(K) peptide from hPANX1 expressed without Src. B ions and Y ions are colored in red and blue, respectively. B, The fragmentation table of the (K)YPIVEQYLKTK(K) peptide shown in panel A. Red and blue color highlighted the B ions and Y ions for the detected amino acid, respectively. The non-phosphorylated Tyr199 is supported by both B ions and Y ions (blue frame). C, The raw spectrum of (K)YPIVEQYLK(T) peptide from human PANX1 expressed with mSrc Y529F mutant. B ions and Y ions are colored in red and blue, respectively. D, The fragmentation table of the the (K)YPIVEQYLK(T) peptide shown in panel C. Red and blue color highlighted the B ions and Y ions for the detected amino acid. The non-phosphorylated Tyr199 is supported by Y ions (blue frame).

Representative LC-MS/MS spectrum covering the Tyr309 for hPANX1.

A, The raw spectrum of (K)VYEILPTFDVLHFK(S) peptide from hPANX1 expressed without Src. B ions and Y ions are colored in red and blue, respectively. B, The fragmentation table of the (K)VYEILPTFDVLHFK(S) peptide shown in A. Red and blue color highlighted the B ions and Y ions for the detected amino acid, respectively. The non-phosphorylated Tyr308 is supported B ions (blue frame). C, The raw spectrum of (K)VYEILPTFDVLHFK(S) peptide from human PANX1 expressed with mSrcY529F mutant. B ions and Y ions are colored in red and blue, respectively. D, The fragmentation table of the the (K)VYEILPTFDVLHFK(S) peptide shown in C. Red and blue color highlighted the B ions and Y ions for the detected amino acid. The non-phosphorylated Tyr309 is supported by both B ions and Y ions (blue frame).

Representative LC-MS/MS spectrum covering the Ser385 for hPANX1.

A, The raw spectrum of (K)TPMSAEMR(E) peptide from hPANX1 expressed without Src. B ions and Y ions are colored in red and blue, respectively. B, The fragmentation table of the (K)TPMSAEMR(E) peptide shown in A. Red and blue color highlighted the B ions and Y ions for the detected amino acid, respectively. The non-phosphorylated Ser385 is supported by both B ions and Y ions (blue frame). C, One raw spectrum of (K)TPMSAEMR(E) peptide from human PANX1 expressed with Src-Y529F mutant. B ions and Y ions are colored in red and blue, respectively. D, The fragmentation table of the (K)TPMSAEMR(E) peptide shown in C. Red and blue color highlighted the B ions and Y ions for the detected amino acid. The phosphorylated Ser385 is supported by Y ions (blue frame). E, One raw spectrum of (K)TPMSAEMR(E) peptide from hPANX1 expressed with Src-Y529F mutant. B ions and Y ions are colored in red and blue, respectively. F, The fragmentation table of the (K)TPMSAEMR(E) peptide shown in E. Red and blue color highlighted the B ions and Y ions for the detected amino acid. The non-phosphorylated Ser385 is supported by both B ions and Y ions (blue frame).

The anti-PANX1 antibody detects both hPANX1 and non-specific proteins.

The non-transfected HEK293T cells or cells transfected with PANX1 (without the GFP tag) is digested by PNGase F and analyzed by western blot. The anti-PANX1 antibody produced two bands from the non-transfected HEK293T cell lysate at approximately 100 kDa and 50 kDa. After PNGase F treatment, the 50 kDa band (indicated by blue dotted lines) can be partially shifted to a location where monomeric PANX1 protein is located. The 100 kDa band (indicated by dotted magenta lines) is not sensitive to PNGase F treatment. The right panel showed a long exposure image of the same blot on the left.

Figure 6—figure supplement 1—source data 1: Uncropped immunoblots for Figure 6— figure supplement 1.

Figure 6—figure supplement 1—source data 2: Raw image for anti-PANX1 immunoblot in Figure 6—figure supplement 1 (left panel).

Figure 6—figure supplement 1—source data 3: Raw image for anti-PANX1 immunoblot in Figure 6—figure supplement 1 (right panel).